Determining DNA sequences by mass spectrometry

ABSTRACT

This invention relates to the methods, apparatus, reagents and mixtures of reagents for sequencing natural or recombinant DNA and other polynucleotides. In particular, this invention relates to a method for sequencing polynucleotides based on mass spectrometry to determine which of the four bases (adenine, guanine, cytosine or thymine) is a component of the terminal nucleotide. In particular, the present invention relates to identifying the individual nucleotides by the mass of stable nuclide markers contained within either the dideoxynucleotides, the DNA primer, or the deoxynucleotide added to the primer. This invention is particularly useful in identifying specific DNA sequences in very small quantities in biological products produced by fermentation or other genetic engineering techniques. The invention is therefore useful in evaluating safety and other health concerns related to the presence of DNA in products resulting from genetic engineering techniques.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to the field of the determination of DNAsequences and the uses of automated techniques for such determinations.

The ability to sequence DNA has become a core technology in molecularbiology, and is one of the great achievements of the last decade. Theease with which sequencing may be accomplished will substantially affectthe rate of development of new drugs produced by recombinant technology,the creation of new biosynthetic pathways and the promise of geneticengineering through manipulation of microorganisms, plants and mammaliancells, and the better understanding of a wide range of diseaseprocesses.

Initially, researchers focused on reading the genetic code and thetranslation of the nucleotide sequence into protein amino acid sequence.This occurs by a process of DNA transcription into mRNA, and then actualsynthesis of the protein on ribosomes. In eucarotic cells, largespecific segments of mRNA, introns, may be excised during anintermediary processing step. Much of the chromosomal DNA is nottranslated, and its specific function is largely unknown. This "excess"DNA was first thought to be excess genetic material. However, asbiologists begin to unravel the details of cell differentiation and theprocesses controlling gene transcription it is now felt that thespecific sequence of these large portions of untranslated DNA may alsoprovide important regulatory signals.

The potential applications which derive from DNA sequencing have onlybegun to be explored. On large scale, analysis of human chromosomal DNAis considered vital to understanding genetic diseases, AIDS and cancerbecause only subtle differences exist between normal DNA and DNAinvolved in pathological conditions. Serious consideration is now beinggiven to the sequencing of the entire human genome--approximately 3billion base pairs. The success of this project will depend on rapid,sensitive, inexpensive automated methods to sequence DNA.

The fundamental approach to determination of DNA sequence has been wellestablished. Restriction endonucleases are employed cleave chromosomalDNA into smaller segments, and recombinant cloning techniques are thenused to purify and generate analyzable quantities of DNA. The specificsequence of each segment can then be determined by either theMaxam-Gilbert chemical cleavage, or preferably, the Sanger dideoxyterminated enzymatic method. In either case, a set of all possiblefragments ending in a specific base are generated. The individualfragments can be resolved electrophoretically by increasing molecularweight, and the sequence on the original DNA segment is then derived byknowing the identity of the terminal base in each fragment.

In its broadest aspect, this invention is directed to methods andreagents for sequencing DNA and other polynucleotides. In particular,this invention described reagents and methods for automating andincreasing the sensitivity of both the Sanger and Gish and Ecksteinprocedures for sequencing polynucleotides. The methods of the presentinvention are based on mass spectrometric determination of the fourcomponent terminal nucleotide residues, where the information regardingthe identity of the individual nucleotides is contained in the mass ofstable nuclide markers.

B. Summary Of The Prior Art

In the Sanger dideoxy method (Proc. Nat. Acad. Sci., 74, 5463 (1977)),the DNA to be sequenced is exposed to a DNA polymerase, a cDNA primer,and a mixture of the four component deoxynucleotides, plus one of thefour possible 2,3-dideoxy nucleotides. There is a competition forincorporation of the normal deoxy- and the dideoxy- nucleotide by thepolymerase into the growing complementary chain. When a dideoxynucleotide is incorporated, further chain extension is prevented. Sincethere is a finite probability that this chain terminating event mayoccur at each complementary site of the appropriate base, a mixture ofall possible fragments ending in that dideoxy base will be generated.This mixture of fragments can be separated by size on gelelectrophoresis. When the experiment is repeated with each dideoxy base,four mixtures of fragments, each terminating in a specific residue areproduced. When this set of mixtures is chromatographed in four adjacentlanes, so that fragment lengths in the four mixtures can be correlatedwith each other, the sequence of the original DNA is determined byrelating the fragment length to the identity of the terminating dideoxybase.

The position of the fragment in gel electrophoresis is usually revealedby staining or by autoradiography. In autoradiography methods, thefragments have typically been labeled with ³² P or ³⁵ S radionuclideswhere either the DNA primer or one of the component deoxynucleotideshave been tagged, and that label incorporated in a specific or randomfashion. The potential number of residues which can be sequenced by thismethod is limited by the experimental ability to correlate the resultsof the four chromatograms (electrophoretograms).

An alternate method of detection was developed by the CaliforniaInstitute of Technology group (Smith, et al., Nature, 321, 674 (1986))in which the identity of the terminal base residues is contained in afluorescent marker attached to the DNA primer. If four fluorescentmarkers of different spectral emission maxima are used, then the fourseparate sets of polymerase fragments can be combined andco-chromatographed. This method is also disclosed in EPO Patent No.87300998.9.

A second variation of the fluorescent tagging approach has recently beenreported by the DuPont group (Science, 238, 335 (1987)) wherein a uniquefluorescent moiety is attached directly to the dideoxy nucleotide. Thismay represent as improvement over the CalTech primer tagging approach inthat a single polymerase experiment can now be run with a mixture of thefour dideoxy terminating bases. However, one trade-off for thissimplification is potential transcription errors by the polymerase,arising from mis-incorporation of the modified dideoxynucleotide baseanalogs.

These modified Sanger methods are an improvement over the originalSanger method in the extent to which DNA an be sequenced because thechromatographic ambiguities have been reduced. However, a number oflimitations are associated with the use of fluorescent labels in thesemodified Sanger reactions. In particular, there are chromatographicdifferences among fragments arising from the unique mobilities of thedifferent organic fluorescent markers. Moreover, there is an inabilityto distinguish individual fluorescent markers because of overlap intheir spectral bandwidths. Finally, there is a low sensitivity ofdetection inherent in the extinction coefficients of the fluorescentmarkers.

All of the above variants of the Sanger method of sequencing have usedslab gel electrophoresis to effect separation of the DNA fragments. Thecasting and loading of slab gels is a skilled but intrinsically manualoperation. The only aspect of this process which has been automated withany success are those commercial devices which read the gel with sometype of laser scanner/spectrophotometer.

Eckstein and Goody, Biochemistry, Vol. 15, No. 8, p. 1685 (1976),discloses a method of chemical synthesis foradenosine-5'-(O-1-thiotriphosphate) andadenosine-5'-(O-2-thiotriphosphate).

Eckstein, Accounts Chem. Res., Vol. 12, p. 204, (1978), discloses agroup of phosphorothioate analogs of nucleotides.

Maxam and Gilbert, methods Enzym., 65:499-500 (1980), disclosed a methodfor DNA sequencing using chemical cleavage. In this method, each end ofa nucleotide to be sequenced is labeled. This nucleotide sample is thenbroken preferentially at one of the nucleotides, under conditionsfavoring one break per strand. This procedure is then repeated for eachof the other three nucleotides. The four samples are then run side byside on an electrophoretic gel. Autoradiography identifies the positionof a particular nucleotide by the length of the fragments produced bycleavage at that particular nucleotide. This method suffers from thesame drawbacks as the Sanger method by requiring long periods ofautoradiography and restricting the length of fragments which can besequenced.

Gish and Eckstein close an alternative method for sequencing DNA and RNAemploying base specific chemical cleavage of phosphothioate analogs ofthe nucleotides which were incorporated in a cDNA sequence. Science,240, 1520-1522 (1988).

Ornstein, et al., Biotechniques, Vol. 3, No. 6, p. 476 (1985), disclosesthe advantages of using ³⁵ S nucleotides rather than ³² P labelling insequencing DNA.

Japanese Patent No. 59-131,909 (1986), discloses a nucleic aciddetection apparatus which detects nucleic acid fragments which areseparated by electrophoretic techniques, liquid chromatography, or highspeed gel filtration. Detection is achieved by utilizing nucleic acidsinto which S, Br, I, or Ag, Au, Pt, Os, Hg or similar metallic elementshave been introduced. These elements are generally absent in the naturalnucleic acids. Introduction of one of these elements into a nucleotideon a nucleic acid allows that nucleic acid or fragment thereof to bedetected by means of atomic absorption, plasma emission or massspectroscopy. However, this reference does not anticipate anyapplication of the described methods or apparatus to the sequencing ofDNA, such as by the Sanger method. Specifically, it does not teach thata plurality of specific isotopes may be used to identify the specificterminal nucleotide residues. Nor does it teach that by total combustionof DNA to oxides of carbon, hydrogen, nitrogen and phosphorus, thedetection sensitivity by mass spectrometry for trace elements, such assulfur which is not normally found in DNA, is vastly improved. Thecombustion step, which is one aspect of the present application, isessential to eliminate the myriad of fragment ions from DNA. Thesefragment ions would normally mask the presence of trace ions of SO₂ inconventional mass spectrometry.

What this reference does disclose is that DNA may be tagged (byundisclosed means) with trace elements, including sulfur, as an aid todetection of DNA, and that these trace elements may be detected by avariety of means, including mass spectrometry.

Details of DNA sequencing are found in Current Protocol In MolecularBiology, John Wiley & Son, Ny., N.Y. Edited by F. M. Ansubel, et al.,1978, Chapter 7 which is incorporated herein by reference. Smith, etal., Anal. Chem., 1988, 60, 438-441, described capillary zoneelectrophoresis--mass spectrometry using an electrospray ionizationinterface and is incorporated herein by reference.

SUMMARY OF THE INVENTION

This invention relates to methods, apparatus, reagents and mixtures ofreagents for sequencing DNA, DNA fragments, and other polynucleotides,whether naturally occurring or artifically made. In particular, thisinvention relates to a method for sequencing polynucleotides based onmass spectrometry to determine which of the four bases (adenine,guanine, cytosine or thymine) is a component of the terminal nucleotide.Specifically, the present invention relates to identifying theindividual nucleotides in a DNA sequence by the mass of stable nuclidemarkers contained within either the dideoxynucleotides, the DNA primer,or the deoxynucleotides added to the primer. This invention isparticularly useful in identifying specific DNA sequences in very smallquantities in biological products produced by fermentation or othergenetic engineering techniques. The invention is therefore useful inevaluating safety and other health concerns related to the presence ofDNA in products resulting from genetic engineering techniques.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a schematic diagram of a complementary DNA sequenceattached to a primer DNA sequence and a typical series of chainterminated polynucleotide fragments prepared according to Scheme Eherein.

FIG. 2A shows in combination a column for separating DNA sequencesaccording o size and a means for sequentially transporting DNA sequencesto a mass spectrometer.

FIG. 2B shows superimposed "ion current vs. time" printouts for ³² SO₂,³³ SO₂, ³⁴ SO₂, and ³⁶ SO₂ resulting from combustion of a chainterminated DNA sequence.

BRIEF DESCRIPTION OF THE INVENTION

This invention relates to methods, reagents, apparatus and intermediatesinvolved in the determination of natural or artifically made("recombinant") DNA sequences and fragments thereof. In particular, thisinvention involves the determination of DNA sequences using acombination of chain termination DNA sequencing techniques and massspectroscopy. Thus, in a typical chain terminating DNA sequencingdetermination such as taught by Sanger, et al., Proc. Nat. Acad. Sci.,74, 5463, (1977) involving a DNA primer, deoxynucleotidetriphosphates,dideoxynucleotidetriphosphates in the presence of a DNA polymerase, suchas the Klenow fragment, are used to determine the DNA sequence. However,in embodiments of the present invention the DNA primer, thedeoxynucleotides or the dideoxynucleotides are labeled with isotopesdetectable by mass spectrometry to determine the DNA sequence. Forexample, if the dideoxynucleotides (A, G, C, T) triphosphates,abbreviated as ddATP, ddGTP, ddCTP and ddTTP respectively, are labeledwith isotopes of different masses respectively, and chain terminatedfragments corresponding to those fragments are separated and analyzed bymass spectrometry, a direct reading of the DNA sequence is obtained.Generally, the labeled component of each dideoxynucleotide component ofthe chain terminated DNA sequence is converted to a more convenientspecies for mass spectrometry determination i.e. sulfur isotopes areoxidized to sulfur dioxide. If the DNa primer or deoxynucleotides arelabeled, reactions between specifically labeled deoxynucleotides must befirst carried out in the presence of a specific dideoxynucleotide. Thisis necessary so that a specific label is associated with a specificchain terminated DNA sequence. Once the individual reactions areconducted, the chain terminated DNa sequences can be mixed, separated,and analyzed by mass spectrometry because there will then be a specificrelationship between a specific isotope and the terminaldideoxynucleotide. This invention is especially useful in determiningthe sequence of small quantities of DNA which are contaminants inproducts resulting from fermentation and other biotechnology relatedprocesses, i.e. "screening". The invention also includes reagents andanalytical instruments for carrying out the above methods as well asintermediate mixtures of chain terminated DNA sequences produced whilecarrying out the methods of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to an improved method for sequencingpolynucleotides using mass spectrometry to determine which of the fourbases (adenine, guanine, cytosine or thymine) is a component of theterminal nucleotide. In particular, the present invention relates toidentifying the individual nucleotides in a DNA sequence by the mass ofstable nuclide markers contained within either the dideoxynucleotides,the DNA primer, or the deoxynucleotides added to the primer. Theinvention also includes reagents and analytical instruments for carryingout the above methods as well as mixtures of chain terminated DNAsequences.

Information regarding the identity of the terminal base in a particularfragment may be signified by using a unique isotopic label for each ofthe four bases. The determination of which isotope marker, and thuswhich terminal base a fragment contains, can then be readilyaccomplished by mass spectral methods. Detection of ions by mass spectrais perhaps the most sensitive physical method available to theanalytical chemist, and represents orders of magnitude bettersensitivity than optical detection of fluorescence.

If stable isotopes are chosen for labeling, then the isotope ratios arefixed by the mode of synthesis. The group of suitable atomic ("nuclide")markers include those from carbon (¹² C / ¹³ /C), chlorine (³⁵ Cl / ³⁷Cl), bromine (⁷⁹ Br / ⁸¹ Br) and sulphur (³² S / ³³ S/ ³⁴ S / ³⁶ S).Since sulfur, chlorine and bromine are not normal constituents of DNA,i.e. they are "foreign", analysis for those foreign isotopes does notrequire consideration of their natural abundance ratio. It is noted thatsulphur is unique among this group in that is alone contains four stableisotopes, each of which can be used to represent one of the fournucleotide bases.

Further, if the fragments are subjected to combustion, then a lightvolatile derivative of the marker atom can be detected. Combustionconverts DNA to the oxides of carbon, hydrogen, nitrogen and phosphorus.The inclusion of a combustion step enormously simplifies the detectionof trace atoms because it eliminates the problems of producing andanalyzing high mass molecular ions.

With sulphur, combustion of the polynucleotide fragments in ahydrogen-oxygen flame or pyrolysis tube will yield sulphur dioxide(SO₂). Thus, assignment of fragment terminal base identity reduces dodetermination of the mass of the SO₂ ion as 64, 65, 66, or 68. This is asimple distinction by existing mass spectral devices using eitherquadrupole or permanent magnet analyzers. For a permanent magnet device,a set of four permanently fixed ion detectors can be mounted tocontinuously monitor the individual ion currents. A quadrupole analyzerwith a single ion-multiplier detector is presently preferred.

These are numerous ways in which a marker isotope could be incorporatedinto the complementary DNA fragment. These include substituting themarker isotope on the pyrimidine, purine, or ribose moieties, or thephosphate bridges between individual nucleotides. Further, the markerisotope may be contained in either part of the cDNA primer, randomlyincorporated along the chain in one or several of the deoxy-base units,or specifically in the terminal dideoxy residue. The only restriction isthat the particular substitution be unique for that particular set offragments.

The site for the stable sulfur label is most preferably the phsophatebridge, using labeled thiophosphate in place of ordinary phsophate. Thetechnique for inserting a stable thiophosphate label in place ofordinary phosphate is similar to that employed in conventional ³⁵ Sradiolabeling experiments. The chemistry and enzymology of thepolymerase reaction using deoxynucleotide α-thiotriphosphates have beeninvestigated extensively. Any future developments in cloning vectors orpolymerase enzymes should also be able to utilize the thiosphosphatederivatives of the present invention.

If the isotope label is to be incorporated into the cDNA primer orrandomly along the chain as a deoxy-base surrogate, then it is necessaryto perform a separate polymerase experiment with each of the appropriatedideoxy-base residues prior to mixing and chromatography. The advantageof using primer or intra-chain labeling is that several atoms of themarker isotope may be incorporated per mole of DNA fragment, and thusenhance detection sensitivity.

If, on the other hand, the isotope label is contained in the dideoxybase itself, then it is not necessary to perform individual polymeraseexperiments. Instead, a mixture of the four dideoxy bases, each with aunique isotope label, together with a mixture of the four normal deoxybases in stoichiometric ratios appropriate for the specific polymeraseenzyme could be used to generate the complete set of labeled fragmentsin a single polymerase experiment. Each fragment, regardless of itssize, will contain one atom of the marker isotope on its terminal(dideoxy) nucleotide, wherein the marker isotope would indicate theidentity of the terminal nucleotide.

Schemes A and B below illustrate typical sulfur and halogen labelingrespectively,

wherein an asterisk (*) is used herein to indicate the presence of anisotopic label in accordance with the present invention;

wherein by A, T, G, and C is meant the bases adenine, thymine, guanine,and cytosine respectively;

wherein by "Alk" is meant straight or branched chain lower alkyl of 1-6carbon atoms;

wherein by "S*" is meant a sulfur isotope of the group of ³² S, ³³ S, ³⁴S and ³⁶ S with the proviso that each isotope be uniquely associatedwith a member of the group consisting of A, T, G, and C respectively;and

wherein by "X*" is meant a "halogen" isotope of the group consisting of³⁵ Cl, ³⁷ Cl, ⁷⁹ Br and ⁸¹ Br with the proviso that each isotope beuniquely associated with a member of the group consisting of A, T, G andC respectively. ##STR1##

Labeling Schemes C, D, and E below show three ways in which specificisotopes, designated as *1, *2, *3, and *4, can be uniquely associatedwith specific terminal nucleotides in a terminated complementary DNA(cDNA) sequence. For convenience in Schemes C-E, the "TP" designationfor the deoxy and dideoxy triphosphates has been deleted. In the firsttwo Schemes, (C and D), the dideoxy chain terminating reaction isconducted separately and then the terminated chains are mixed prior toseparation. In particular, Scheme D is a modification of the chemicalcleavage procedure of Gish and Eckstein, Science, 240, 1520 (1988)whereby the DNA fragment is selectively cleaved by base adjacent thephosphothioate linkage, leaving a labeled deoxy compound as the terminalnucleotide in the fragment. In Scheme D, one creates a series of suchfragments which differ from one another solely in size, via the presenceof an additional terminal nucleotide. Identification of each terminalnucleotide (via their isotopic marker) in relation to size of thefragment provides the base sequence of the DNA or polynucleotide ofinterest.

In Scheme E, a mixture of the four individually labeled dideoxynucleotide triphosphates, together with a mixture of the four deoxynucleotide triphosphates are reacted together with the primer ("P") in asingle reaction. Because only one

C Labeled Primers

    ______________________________________                                        1. P.sub.A*1 + d(A,C,G.T) + dd(A)                                                                →                                                                             P.sub.A*1 dN.sub.-- dN . . . dd(A)                  2. P.sub.C*2 + d(A,C,G.T) + dd(C)                                                                →                                                                             P.sub.C*2 dN.sub.-- dN . . . dd(C)                  3. P.sub.G*3 + d(A,C,G.T) + dd(G)                                                                →                                                                             P.sub.G*3 dN.sub.-- dN . . . dd(G)                  4. P.sub.T*4 + d(A,C,G.T) + dd(T)                                                                →                                                                             P.sub.T*4 dN.sub.-- dN . . . dd(T)                  ______________________________________                                    

D. Labeled Deoxy

    __________________________________________________________________________    1. P + d(A,C,G,T) + d(A*.sup.1) + dd(A) →                                                    P . . . dN.sub.-- dA*.sup.1 dN . . . dd(A)              2. P + d(A,C,G,T) + d(C*.sup.2) + dd(C) →                                                    P . . . dN.sub.-- dC*.sup.2 dN . . . dd(C)              3. P + d(A,C,G,T) + d(G*.sup.3) + dd(G) →                                                    P . . . dN.sub.-- dG*.sup.3 dN . . . dd(G)              4. P + d(A,C,G,T) + d(T*.sup.4) + dd(T) →                                                    P . . . dN.sub.-- dT*.sup.4 dN . . .                    __________________________________________________________________________                          dd(T)                                               

E. Labeled Dideoxy

    __________________________________________________________________________    1. P + d(A,C,G,T) + dd(A*.sup.1,C*.sup.2,G*.sup.3,T*.sup.4)                                         P . . . dN.sub.-- dN . . . dd(A*.sup.1)                                       P . . . dN.sub.-- dN . . . dd(C*.sup.2)                                       P . . . dN.sub.-- dN . . . dd(G*.sup.3)                                       P . . . dN.sub.-- dN . . . dd(T*.sup.4)                 __________________________________________________________________________

reaction and one separation need be run in Scheme E, it can be readilyseen that the labeled dideoxy scheme, Scheme E, is the preferred methodof the present invention. Particularly preferred reactants in Scheme Eare the labeled dd(A*,C*,G*, or T*) triphosphates where the labels ³² S,³³ S, ³⁴ S and ³⁶ S replace a phosphate oxygen as shown in Scheme A.

Accordingly, the invention not only includes reagents but also a mixtureof unique isotopically labeled dideoxynucleotide triphosphates (ddC*TP,ddG*TP, ddT*TP and ddA*TP) where each dideoxynucleotide triphosphate islabeled with a different sulfur or halogen isotope. In particular,sulfur labeling, consisting of the isotopes ³² S, ³³ S, ³⁴ S and ³⁶ S,is preferred.

The invention also includes the intermediate mixture of dideoxy chainterminated DNA sequences where each chain terminated DNA sequencecontains an isotope measurable by mass spectrometry which is related toa specific chain terminating dideoxynucleotide. Considering thedideoxynucleotide chain terminated DNA sequence to have the followingsections: DNA primer--deoxynucleotide segment--chain terminatingdideoxynucleotide, either section of the chain terminated DNA sequencecan be labeled, provided the labeling procedure associates a specificlabel with a specific chain terminating dideoxynucleotide. The inventionalso includes the mixture of isotope labeled chain terminated DNAsequences separated by size.

Although this invention has been discussed in terms of sequencing DNA,there is no reason to believe that it could not also be used to sequenceRNA by substituting a unique isotopically labeled uridine nucleotidetriphosphate, UTP, in place of the deoxythymine nucleotide triposhpated(T)TP, discussed herein.

FIG. 1 illustrates a complementary DNA sequence attached to a promoterDNA sequence and typical series of chain terminated polynucleotidefragments prepared according to Scheme E from mixtures ofdeoxynucleotide triphosphates and labeled dideoxy nucleotidetriphosphates. These labeled fragments illustrate labeled chainterminated complementary DNA sequences 1 prepared by the method of thepresent invention, wherein the size of each complementary DNa fragmentcorresponds to the relative position of that fragment's terminalnucleotide in the overall complementary DNA sequence. These labeledfragments sequences are separated by size by an electrophoresis column2. The fragments from the electrophoresis column 2 are sequentiallyeluted to a detector 3. FIG. 2A shows in more detail the apparatus fordetermining a DNA sequence. DNA sequences are prepared in reactionchamber 4. The mixture of labeled terminated DNA fragments are separatedaccording to size by electrophoreses on a polyacrylamide gel column 5wherein migration occurs from the cathode (V³¹) to the anode (V³⁰). thefractions are taken off the polyacrylamide gel column 5 sequentially bysize at transfer point 6 where there is provided a means 7 fortransferring the terminated DNA fragments to an oxidizer or combustionchamber 8. In the oxidizer or combustion chamber 8, the sulfur label isoxidized to SO₂ and the labeled SO₂ is detected in a mass spectrometer9. FIG. 2B shows typical superimposed "ion current v. time" plots form/e 64, 65, 66, and 68, corresponding to ions produced by the fourstable isotopes of sulfur, i.e., ³² SO₂, ³⁴ SO₂, and ³⁶ SO₂,respectively. When the stable isotopes of sulfur associated with thebases, A, C, G and T are ³² S, ³³ S, ³⁴ S, and ³⁶ S respectively, a plotcorresponding to the DNA sequence illustrated at the top of FIG. 2B isobtained. In this manner, the DNA sequence of any genetic material canbe determined automatically and on femto- to nanomolar quantities ofmaterial.

There are several variations in the design of an automated DNA sequencerof the present invention. The major components of the device are thereaction chamber for conducting polymerase reactions, thechromatographic device consisting of some form of electrphoresis, theeffluent transport, the combustion system, and the mass spectralanalyzer. Because this instrument is designed to operate on nano- tofemto-molar quantities of DNA, it is important that the geometry of allcomponent systems be kept to a minimum size.

The chromatographic system may be of a laned plate or tubularconfiguration. In the plate designs, the supporting medium for thechromatogrpahic separation will be most preferably a polyacrylamide gel,where the ratio of acrylamide to bis-acrylamide is more preferablybetween 10:1 and 100:1. Although persulfate is the typicalpolymerization catalyst used by most workers, the background of sulfateions may be unacceptably high without extensive washing. Sodiumperborate or ultraviolet irradiation can be used successfully toinitiate cross-linking and produce high quality gels, which can be usedimmediately without washing.

For tubular designs, the chromatographic separation may be conducted ina gel-filled capillary or in an open tubular configuration. Thepreferred dimensions of the capillary depend on whether an open orgel-filled medium is selected. For gel-filled devices, preferreddiameters are 50 to 300 microns. In open tubular configurations,however, the preferred diameters are 1 to 50 microns. The preferredlength of the capillary depends on the diameter and the amount of DNAsample which will be applied, as well as the field strength of theapplied electrophoretic voltage. The preferred length is optimallybetween 0.25 and 5 meters.

For open tubular configurations, the capillary will preferably befabricated from fused silica. Under typical operating conditions, wherethe pH of the buffer is usually maintained in a range of 5.0 to 11.0,the surface of the silica will have a net negative charge. This surfacecharge establishes conditions in which there is a bulk electroosmoticflow of buffer toward the negative electrode. The DNA fragments alsopossess a negative charge and are attracted to the positive electrode.Hence, they will therefore move more slowly than the bulk electroosmoticflow. In gel-filled devices, the supporting medium minimizeselectroosmosis. Since the gel has no charge, the negatively charged DNAfragments migrate toward the positive electrode.

There are a variety of techniques to modify the surface charge on thewall of an open capillary. One particularly useful method is tocovalently modify the wall with a monomer such asmethacryloxypropyltrimethoxysilane. This monomer can then be crosslinkedwith the acrylamide to give a thin bonded monolayer which is similar incharacteristics to the polyacrylamide gel-filled capillaries. Thedistinct advantage of the coated wall method is that the capillary canbe recycled after each analysis run simply by flushing with freshbuffer. (See S. Hjerton, J. Chromatography, 347, 191, (1985) fordetails.)

The transfer system is selected to match the particular chrmoatographicdesign. The chromatographic system may be of laned plate or tubularconfiguration. It is desirable to have the chromatography effluent in aclosed environment. The tubular configurations may be more amenable tosample transfer designs which pump, spray or aspirate the columneffluate into the combustion chamber, and thus minimum degradation inresolution because of post-chromatographic remixing of fractions.

For the open plate type devices, a moving belt or wire system can beused satisfactorily. In this system, a thin coating of the columneffluent is spread on the ribbon to transport the eluted fractionsthrough a pre-drying oven and then into the combustion furnace. Thetransport ribbon may be fabricated from platinum or other nobel metal,and may be continuously looped because the ribbon can be effectivelycleaned upon passage through the combustion furnace. Less preferably,the ribbon may be fabricated from a glass or ceramic fiber or carbonsteel. In this design, the ribbon would be taken up on a drum fordisposal.

For tubular configurations, the most preferred embodiment of thetransfer system is to use an electrospray nebulizer method to create afine aerosol of the column effluent. In this technique, a small chargeof optimally less than 3000 volts is applied to the emerging droplet.The charge of a larger droplet tends to disperse it into a very finemist of singly charged droplets. These fine charged droplets can befocused and directed by electric or magnetic fields, much as in anink-jet printer head. It is important to control the temperature of theflowing gas stream into which the aerosol is introduced. If thetemperature is too great, then the droplets will tend to evaporate onthe capillary injector. If the temperature is too low to overcome thesurface tension of the effluent, the individual droplets will notadequately be dispersed. The composition and ionic strength of thesupporting electrolyte is important. The preferred buffers are phosphateor borate at less than 0.1 M concentration. Because the overall methodis based on detection of trace atoms, it is critically important thatthe buffers be free of contaminant ions, such as sulfate.

A second satisfactory method to create an aerosol of the column effluentis an ultrasonic device which produces sufficient mechanical shear andlocal heating to disperse the droplet. A similar type of shear may alsobe generated off the tip of a capillary injector into a venturri typeaspirator, where the flow of supporting gas is developed by the pressuredifferential into the mass spectrometer. In these designs, it is oftendesirable to add small amounts of additional aqueous or organic solventsat the tip in order to aid in the flash evaporation of the effluent.

The combustion section is designed to completely burn the vaporized orsurface evaporated chromatography effluent of DNA fragments togetherwith the supporting electrolytes and optional solvent modifier to theoxides of carbon, hydrogen, nitrogen, phosphorous, and most importantly,those of the marker isotope. The sensitivity of detection of the markerisotope is greatly affected by the efficiency of combustion, since lowmolecular weight fragment ions which may result from incomplete burningcan mask the presence of the primary detection species. The specificmass/charge range which is must be free of interfering ions for sulfurdioxide detection is 64 to 68.

This combustion may be accomplished at moderate to approximatelyatmospheric pressure prior to injection into the mass spectrometer. Forsulfur containing streams, essentially complete combustion to sulfurdioxide will be achieved when the sample is heated to temperatures inexcess of approximately 1200° C. in an oxygen environment.

The most rugged design is to simply aspirate the column effluent into ahydrogen-oxygen flame, similar in design to standard gas chromatographyflame ionization detectors. The important characteristics of the flame,the temperature and sample residence time, will be determined by theratio of hydrogen to oxygen, the aggregate flow rate of gases and thelocal pressure. The characteristics of sulfur containing flames at100-150 tor have been described by Zachariah and Smith, Combustion andFlame, 69, 125 (1987). A limitation to the sensitivity of this design isthe volume of gas (water vapor) resulting from hydrogen-oxygencombustion which effectively dilutes the sulfur dioxide. Although thestandard mass spectrometry techniques such as gas separators orsemipermeable membranes may be used to remove water vapor, there is atrade-off between sample dilution and ultimate detectability which mustbe considered for each design.

A preferred method to very efficiently burn the nebulized columneffluent is to inject it into a short heated tube in an oxygenenvironment. The tube may be constructed of nobel metals such asplatinum, ceramic or quartz, depending on the method of heating. Theexternal heating action may be provided by a cartridge electricalresistance heater or an external flame. The tube may be packed with aheat exchanger medium such as glass wool. Optionally, a catalyticsurface may also be provided by such materials as supported platinum orcopper oxide to enhance combustion efficiency.

A particularly effective method to burn the sample is in an inductivelycoupled oxygen plasma, where the tube forms the resonant cavity of amicrowave generator. The inductively coupled plasma techniques have beenreviewed by G. Meyer, Anal. Chem., 59, 1345A (1987).

Alternatively, the combustion may be effected within the low pressureenvironment of the mass spectrometer. A standard ionization technique inmass spectrometry is fast atom bombardment. An energetic beam of atoms,usually xenon, is produced in a plasma torch and directed toward thesample. Ionization occurs by collision induced dissociation of this beamwith the sample. If instead of pure xenon, oxygen is introduced into thefast atom beam, then both oxidation and ionization of the sample canoccur. The limitation of this method however is the difficulty inachieving quantitative oxidation, and minimizing the background signalfrom incompletely oxidized low mass fragments.

There are several methods to effect ionization of sulfur dioxide. Theobjective is to obtain as high ion current as possible. In designs whichoperate at atmospheric pressure by flame, corona discharge needle ormicrowave induced plasma discharge, the ionization efficiency will bevery high. In this type of design, a portion of the ionized gas inintroduced into the low pressure region of the mass analyzer through asmall sampling orifice or skimmer cone. The size of the orifice, andthus the percentage of total combustion sample which can be introduced,will depend on the pumping speed of the vacuum system. Generally, lessthan five percent of the sample will be transferred to the analyzerregion. Designs of this type have been described by T. Covey, Anal.Chem., 58, 1451A (1986) and by G. Hieftje, Anal. Chem., 59, 1644 (1987).

Alternatively, the sample may be ionized in the lower pressure regionnear the analyzer. This may be achieved by such methods as electronimpact using the beam emanating from a hot filament, by fast atombombardment with inert gases such as xenon, or by chemical ionizationwith a variety of light gases. In these type of design, although theionization efficiency is low relative to atmospheric methods, a greaterpercentage of those ions actually get to the analyzer section. Theelectron impact techniques have been described by A. Brandy, Anal.Chem., 59, 1196, (1987).

An RF-only quadrupole mass filter may be used to help separate lowmolecular weight combustion products (H₂ O, N₂ and CO₂).

The analyzer and ion detector sections can be selected from severalcommercially available designs. The analyzer may be a quadrapole devicewhere mass selection depends on the trajectory in a hyperbolic field, afield swept electromagnetic device with a single ion detector, or apermanent magnet device with an array of four ion detectors tuned to theisotopes of interest. The detector may be of single stage or ionmultiplier design, although the latter type is preferred for highestsensitivity.

When the mass spectrometer is being used to detect isotopes of sulfurdioxide, as would be the case when dideoxy terminated thiophosphates arebeing utilized, the highest sensitivity is achieved when the polarity ofthe spectrometer is set to determine the positive ion spectrum.

When the mass spectrometer is being used to detect isotopes of chlorineor bromine, then maximum sensitivity will be achieved when thespectrometer polarity is reversed, and the negative ion spectrum isdetected.

The labeled compounds of the present invention are prepared byconventional reactions employing commercially available isotopes. Forexample, the sulfur isotopes: ³² S, ³³ S, ³⁴ S and ³⁶ S are commerciallyavailable as CS₂ or H₂ S from the Department of Energy (Oak Ridge, Tenn.or Miamisburg, Ohio) at "isotopic enrichments" of 99.8%, 90.8%, 94.3%and 82.2% respectively. Although the method of the present inventionwill provide satisfactory results with the "isotopically enriched"commercial products, it is preferred that the sulfur isotopes be at99.5% enrichment to facilitate interpretation of the ion current v. timeplots.

Enrichment techniques for sulfur are well known in the art. Inparticular, CS₂ can be further enriched by fractional distillation whichtakes advantage of the different boiling points of CS₂ conferred by thevarious sulfur isotopes. Alternatively, gaseous diffusion of SF₆ alsocan provide further enrichment of the sulfur isotope of interest.Thereafter, the isotopically enriched CS₂ *, H₂ S* or SF*₆ are convertedinto the reagents described herein by techniques well known in the art.

The "halogen" isotopes ³⁵ Cl, ³⁷ Cl, ⁷⁹ Br, and ⁸¹ Br are alsocommercially available from the Department of Energy in either elementalform or as the corresponding halide salt at enrichments of 99%, 95%, 90%and 90% respectively. These isotopes are used herein in theircommercially available form.

As shown in Scheme F, labeled2',3'-dideoxynucleotide-5-(O-1-thiophosphates) III are prepared byinitially reacting a 2',3'-dideoxynucleotide I with isotopicallyenriched thiophosphoryl chloride (PS*Cl₃) in triethyl phosphate, whereinby "S*" is meant a sulfur isotope that is a member of the groupconsisting of ³² S, ³³ S, ³⁴ S and ³⁶ S in isotopically enriched form.From the above reaction, the correspondingly2',3'-dideoxynucleotide-5'-(O-1-thiotriphosphate) III is prepared fromII by dissolving the bistriethylamine (TEA) salt of II in dioxane andreacting it with diphenyl phosphochloriodate to form the diphenylphosphate ester of II. This phosphate ester was further reacted with thetetrasodium salt of pyrophosphate in pyridine to form III. Purificationof III is accomplished by chromatography on diethylaminoethyl (DEAE)cellulose.

Scheme G shows the general method for preparing3'-halo-2',3'-dideoxynucleosides V wherein the halogen ("X*") is amember of the group consisting of ³⁵ Cl, ³⁷ Cl, ⁷⁹ Br, and ⁸¹ Br inisotopically enriched form. In particular, a solution of1-(5-O-triphenylmethyl-2-deoxy-β-D-threopentofuranosyl)nucleosidewherein by nucleoside is meant A, T, G, or C, in a basic solvent, suchas pyridine, was reacted with methanesulfonyl chloride. The resultingmesylate was treated with an isotopically enriched salt, such as LiX*,in the presence of heat and then acidified to produce a3'-halo-2',3'-dideoxynucleoside V. Compounds of Formula V can beconverted to the corresponding labeled nucleotide monophosphate VI byreaction with cyanoethyl phosphate and dicyclohexylcarbodiimide (DCC)followed by LiOH deblocking.

The corresponding triphosphate of V is prepared from the monophosphateas described from the monophosphate as described for the conversion ofII to III above.

Scheme H present a method for halogenating a purine or pyrimidine baseof a 2', 3'-dideoxynucleoside VII using isotopically enriched elementalbromine (⁷⁹ Br or ⁸¹ Br) or chlorine (³⁵ cl or ³⁷ Cl), i.e. X₂ *. The2', 3'-dideoxynucleoside VII is dissolved in a polar solvent, such asdry DMF, in the presence of a base, such as pyridine. To the reactionmixture is added a molar equivalent of the elemental halogen (X₂ *) andthe reaction mixture is allowed to stir for 12 hours. Evaporation of thesolvent produces the labeled 2',3'-dideoxynucleoside VIII wherein theisotopic halogen label is on the purine or pyrimidine base of thedideoxy nucleoside. Purification is accomplished by conventionalchromatographic techniques.

The labeled 2',3'-dideoxynucleoside VIII is converted to thecorresponding monophosphate and triphosphate as discussed above for VIand III respectively.

The examples described herein are intended to illustrate the presentinvention and not limit it in spirit or scope. ##STR2##

EXAMPLE 1 Preparation of [³²S]2',3'-Dideoxyadenosine-5'-Phosphorothioate

2',3'-Dideoxyadenoisine (47 mg, 0.2 mmol) was suspended in triethylphosphate (0.5 ml) and heated briefly to 100° C. The solution was cooledto 4° C. and treated with [³² S] thiophosphoryl chloride (37 mg, 0.22mmol). The mixture was agitated for 12 hr. at 4° C., and then treatedwith 2 ml 10% barium acetate and agitated at 20° C. for 1 hour. Thesuspension was treated with 0.5 ml triethyl amine and then with 5 ml 95%ethanol. The suspension was agitated 30 min. and then filtered. Theprecipitate was washed with 50% aqueous ethanol and then water. Thefiltrate was evaporated to dryness, and the solid taken up in water andchromatographed on a column of diethylaminoethyl (DEAE) cellulose whichhad been equilibrated with NH₄ HCO₃. The column was eluted with 0.1 MNH₄ HCO₃ and the fractions adsorbing at 260 nm were pooled andevaporated. The solid was evaporated twice with 80% ethanol, twice with80% ethanol containing 2% triethyl amine (TEA), and finally withanhydrous ethanol. There was obtained 44 mg of the bis-triethylaminesalt of the title product. A solution of the triethylamine salt in 1 mlmethanol was treated with 1 ml of a solution of 6 M NaI in acetone. Theprecipitate was washed with acetone and dried to give 32 mg of disodiumsalt of the title product as a white solid.

EXAMPLE 2 Preparation of [³²S]2',3'-Dideoxyadenosine-5'-(O-1-Thiotriphosphate)

A solution of the bis-triethylamine (TEA) salt of the title product ofExample 1 (26 mg, 0.05 mmol) was dissolved in 1 ml dry dioxane andtreated with diphenyl phosphochloridate (0.015 ml, 0.075 mmol). Themixture was agitated for 3 hr. at 25° C. A solution of dry pyrophosphatein pyridine was prepared by dissolving the tetrasodium salt (220 mg, 0.5mmol) in 3 ml pyridine and evaporating twice, and the taking in 0.5 mlpyridine. This solution was added to the above solution of the crudeactive ester, and stirred for 2 hr. The crude product was precipitatedby addition of ether (10 ml). The precipitate was dissolved in water andchromatographed in DEAE-cellulose eluted with 0.1 M triethylammoniumbicarbonate. The pooled fractions contained 150 A₂₆₀ units (20%) of thetitle product. The solution was lypholyzed and the residue stored at-70° C.

EXAMPLE 3 Preparation of [⁷⁹ Br]3'-Bromo-2',3'-Deoxythymidine

A solution of1-(5-O-triphenylmethyl-2'-deoxy-β-D-threopentofuranosyl)thymine (50 mg,0.1 mmol) in pyridine (1 ml) was treated with methanesulfonyl chloride(0.014 ml, 0.12 mmol) and the reaction agitated at 10° C. for 6 hr. Themixture was then evaporated, diluted with CHCl₃ (5 ml) and washed 2×with water. The organic layer was evaporated, and the crude mesylatedissolved in dry diglyme (1 ml) and treated with [Li⁷⁹ Br] (17 mg, 0.2mmol). The solution was heated for 4 hr. at 100° C., and then dilutedwith 80% acetic acid (1 ml) and heated 15 min longer. The reaction wascooled and diluted with water (2 ml), and extracted 3× with chloroform(2 ml). The organic extracts were evaporated and the residuechromatographed on silica gel eluted with 95:5 chloroform methanol togive 18 mg of the title product as an off-white solid.

this material could be converted to the triphosphate via themonophosphate as in Example 2. The monophosphate was prepared byreaction with cyanoethyl phosphate and dicyclohexylcarbodiimide (DCC),followed by LiOH deblocking.

EXAMPLE 4 Preparation of [⁷⁹ Br]2',3'-Dideoxy-5-Bromocytidine

To a solution of 2',3'-dideoxycytidine (60 mg, 0.3 mmol) in dry DMF (1ml) was added 0.1 ml pyridine and then [⁷⁹ Br] bromine (42 mg, 0.3mmol), and the mixture agitated for 12 hr. The solvent was evaporated,and the residue chromatographed on silica gel (ethylacetate:methanol:triethylamine 90:10:1) to give 46 mg of the titleproduct as a white solid.

This material could be converted to the triphosphate via themonophosphate as in Example 2. The monophosphate was prepared byreaction with cyanoethyl phosphate and dicyclohexylcarbodiimide followedby LiOh deblocking.

What is claimed is:
 1. In a process for determining DNA sequence by thedideoxynucleotide chain termination method, the improvement comprisingincorporating ³² S, ³³ S, ³⁴ S and ³⁶ S in the formation of the chainterminated sequence so that a unique sulfur isotope is associated withthe terminal nucleotide in the chain terminated sequence, separating thechain terminated sequences by capillary gel electrophoresis, combustingthe separated chain terminated sequences to convert the incorporatedsulfur to SO₂, and determining the terminal nucleotide by measuring ⁶⁴SO₂, ⁶⁵ SO₂, ⁶⁷ SO₂, and ⁶⁸ SO₂ in a mass spectrometer therebydetermining the sequence of the DNA.
 2. A process according to claim 1wherein the isotope is incorporated only into the 2',3'-dideoxyribonucleotides.
 3. A process according to claim 1 wherein theisotope is incorporated only into the 2'-deoxyribonucleotides.
 4. Aprocess according to claim 1 wherein the isotope is incorporated into aDNA primer.